Hello, everybody and welcome back to the series of lessons on biorisk management. In this second lesson on biosafety cabinets, we will discuss how they work and how to use them. Since the most common type of cabinet is the Class II, A2 cabinet, we will focus on this one only. Do you remember from our previous lesson what a Class II, A2 cabinet protects? First, it protects you by drawing the air away from your breathing zone when you’re sitting in front of the biosafety cabinet. The air moves away from you, in through the sash and into the front cabinet grill. Second, it protects the environment, by exhausting the air that may contain potentially infectious aerosols through a HEPA filter. Third, it protects the product by HEPA filtering the air as it returns to the work surface. 

All practices for how to use a BSC should be outlined in your SOP. The SOP must describe how the cabinet works, how you operate it, who to contact for repairs, what to do if an alarm goes off, who should you should contact, and how to maintain the work area? Once the SOP is created it is important to document that all users are trained and are following the SOP. The cabinet will only protect you if you use it correctly. 

When you start using your cabinet and first approach it, you may find the UV light on (if there is one). Please turn that off and turn on the light. The sash may be blocked or down, which you’ll need to open. Turn on the blower or fan and check the certification date. Here is what a certification sticker may look like. That gives you assurance that the cabinet has been tested recently and that it’s been certified. The BSC should have been certified within the last year. Make sure the sump drain valve (if there is one) is closed. This is the valve at the bottom of the cabinet where the sump is. The sump collects any large spills within the cabinet. Decontaminate all the work surfaces inside the cabinet to make sure they’re clean, with suitable disinfectant. This includes all the surfaces including the glass. Make sure that the sash height is correct (if adjustable) and that there are no error messages, alarms or flashing lights. If there’s a pressure gauge, make sure you check that the reading is within the correct range. All of these procedures should be documented in your standard operating procedure and everyone should have been trained on these procedures. Make sure the cabinet runs for at least five minutes before using it to clean the air inside. Now you are ready to put your supplies inside and start work. 

Here is a picture of the pressure differential gauges. Not all cabinets have these because the new cabinets are now using electronic gauges. These gauges usually measure the pressure drop across the HEPA filter to ensure that it’s not blocked. This gives you some indication of how well the cabinet is working. Again, the newer cabinets now have a lot more monitoring of various air flows and pressure differentials which will alarm if they are out of the acceptable range.
You see in this slide two different workers using the BSC in two different ways. The biggest difference is in their respiratory protection. The person on the left has no respiratory protection, while the person on the right is wearing a powered air purifying respirator. What face and respiratory protection you wear at the BSC is dictated more by the hazards that may be present in the room that you are working in rather than the hazards inside the BSC. A BSC that is functioning well should protect the worker from any infectious aerosols and slashes to the face. However, based upon your risk assessment you may still wish to wear face and respiratory protection.
Watch this movie now from the WHO on biosafety cabinet preparation and how it should be used properly. 

Video

In the previous video you saw some really good work practices on how to set up the biosafety cabinet and how to put in materials. I just want to remind you of a few key points that were discussed. When placing materials in the BSC, try to separate the space into working zones. Separate the clean from your dirty area with a work zone in the middle. Put down a paper towel or a plastic- backed absorbent pad in the middle work zone. Having a pad under the work zone makes it very easy to capture any spills, drops or splashes and clean the cabinet. You also see in the picture a horizontal tray on the far right-hand side for disposal of your long, contaminated pipettes. It is recommended to lay your pipettes down horizontally after they’ve been used either in water or disinfectant bath. Again, organize your work inside the cabinet to ensure clean and contaminated zones do not overlap. 

While working in the cabinet it is very important to minimize any movement that might disrupt the air curtain at the front of the cabinet. Remember, the air moving into the front of the cabinet and the air curtain is what protects you because it draws air away from you and prevents any release of infectious aerosols from coming out of the cabinet. Therefore, if you’re in a small room, keep the doors closed so that there wouldn’t be any disruption of the air curtain from doors opening and closing. In larger rooms, located the cabinet as far back in the room and away from the door. Rapid movements of your hands or arms can also disrupt the air curtain. When moving your arms in and out of the cabinet, move them directly forward away from you and directly backwards towards you and not side-to-side. This is less likely to break the air curtain. In addition, if there’s any spills or small droplets you should clean those up immediately. Again, if you work over an absorbent pad that makes cleaning up spills a lot easier. You can just bundle up the pad and throw it away and put down a new one. 

When working in a BSC, try to minimize the creation of aerosols. This was covered before in the lesson on good work practices. Try to minimize blowing out pipettes, vortexing open tubes, or entering pressurized vessels with uncovered needles. All these actions create splashes, splatters and potential aerosols. Even though the BSC is protecting you from infectious aerosols, still try to minimize the creation of those as much as possible. Be sure not to block the airflow in the BSC when using it. As shown in the picture, air must flow from the supply HEPA filter down to the worksurface and then be drawn into the back and front intake grills. If you put a lot of equipment along the back, you may block the air flow. Try to limit the amount of equipment you must put in the BSC and leave an air space between the back of the equipment and the rear grill. Don’t rest your arms across the front of the grill, which may again block the airflow to the front grill. Do not store materials on top of the biosafety cabinet as you might block the cabinet exhaust. If that grill is blocked and the air cannot go out, then it won’t come into the cabinet either. The BSC is a closed box – whatever comes in must go out. Modern cabinets with air flow sensors will alarm if you do this because the airflow is decreased. Do not use constantly burning flames inside the BSC. Bunsen burners or alcohol jars are commonly used to flame loops and the openings of vessels. This is an old standard microbiological technique to create some sterility around spaces and on objects. This may still be useful if you are working on the open bench where the air may not be clean, but inside the biosafety cabinet the air is very clean and this is not necessary. There may be some times when you need to sterilize or heat certain things. If so, it may be acceptable to use a touch activated flame. These are only on when you touch them providing a small burst of heat for you to sterilize or heat fix something. If you do bacteriology and have loops that need to be sterilized you should use an electric incinerator or disposable loops instead of a flame. You see in the picture, an electric incinerator, where you can put the metal loop inside a heated tube. You should not use constantly burning open flames inside a BSC, because the heat from the flame can damage the HEPA filter and disrupt the air flow inside the cabinet. In addition, if you using ethanol as well, you can have a serious fire that could destroy your cabinet.
This is just a picture of what can happen if you combine a Bunsen burner and alcohol. There was a huge fire inside this biosafety cabinet, which completely destroyed it. We don’t want this happening to you at your workplace.
If you need vacuum for your work, be sure your vacuum pump is protected with traps. This picture shows two bottles in series. The first one will catch any liquids you are aspirating and the second one will catch any overflow. In addition, it is also a good idea to put a HEPA or 0.22 micron filter in the vacuum line to protect the vacuum pump and the environment from any infectious aerosols that might be drawn into the vacuum system. Here’s another great video that shows you some good biosafety cabinet practices and procedures.

Video

When you are finished your work for the day and are ready to leave the cabinet, start by removing all the materials you put inside. Before bringing materials out, be sure to decontaminate the exterior surfaces of all equipment and materials, such as the waste bag and pipette discard pan. Spray everything with a suitable disinfectant, wait for the required contact time and then wipe of any excess liquid. Once the BSC interior is empty of the materials and equipment you can proceed with disinfecting all the interior surfaces, as you did before you started work. Here again is another good video on finishing your work day. Please watch this video.

Video

Many cabinets have ultraviolet (UV) lights installed as a germicidal lamp. UV should not be relied upon as primary means of decontamination inside the BSC. You still need to use a liquid disinfectant for surface decontamination. UV as a decontamination method has several serious limitations that you should be aware of. The lamp intensity and wavelength must be correct in order for it to work properly. As the bulb ages the intensity decreases. Any dirt or dust on the bulb also decreases intensity. As the distance from the bulb increases, the intensity of the UV decreases and there is poor penetration of the microbe being inactivated. Therefore, intensity and wavelength must be validated regularly. UV rays only travel in a straight line. Therefore, any shadows (areas that block UV rays) will not be disinfected. If you do use UV light as a secondary means of decontamination inside the BSC, 15 minutes of use is generally sufficient. More than 15 minutes destroys plastics and the glue in HEPA filter and BSC. Turn on UV only when others are not present. UV light can cause skin and retinal burns if directly exposed. Follow the link provided to learn more.

There is a lot of good information on the internet and in various publications about biosafety cabinet use and certification. NSF 49 is the primary guidance document used by the US, which describes how the cabinets are manufactured, tested and field certified. The Canadian Biosafety Standard, talks about the selection, instillation, and use of biological safety cabinets. The US BMBL also has an appendix that talks more about biosafety cabinets. Please follow the links provided for further information.

In summary, I want to remind you of some of the key points about using a biosafety cabinet property, so that it can protect you, the work product, and the environment. First, purge the cabinet for at least 5 minutes and then clean it before use. Try to eliminate or minimize the use of flames inside the BSC because of the potential fire hazard, disruption to the airflow and potential damage to the HEPA filter. Do not rely upon UV light for disinfection of the interior. Do not overload the biosafety cabinet with equipment and materials. Always disinfect the cabinet with a liquid disinfectant after use. Maintain the biosafety cabinet with good routine procedures and annual certifications. Do routine maintenance on a weekly, monthly and annual basis, as described in your SOPs. Finally, ensure that all the users are trained in the correct biosafety cabinet use and emergency procedures. Take care of it and it will take care of you.
Thank you again for taking this lesson on how to use a biosafety cabinet. The next lesson will explain how biosafety cabinets are certified.